The available SRL3 deletion strain of Saccharomyces cerevisiae contains a truncation of DNA damage tolerance protein Mms2: Implications for Srl3 and Mms2 functions

摘要

A screen of the commercially available collection of haploid deletion mutants of Saccharomyces cerevisiae for spontaneous mutator mutants newly identified a deletion of SRL3. This gene had been previously isolated as a suppressor of lethality of checkpoint kinase deletions if overexpressed. We found DNA damage sensitivity and extended checkpoint arrests to be associated with this strain. However, when crossed to wild-type, a mutant gene conferring these phenotypes was found to segregate from the SRL3 deletion. The mutation was identified as a C-terminal truncation of Mms2, an E2 ubiquitin conjugating enzyme involved in error-free replicative bypass of lesions. This confirmed an earlier report that Mms2 may be required to restrain error-prone polymerase zeta activity and underscored that residues of the C-terminus are necessary for Mms2 function. Srl3, on the other hand, does not appear to influence DNA damage sensitivity or spontaneous mutability if deleted. However, the absence of these phenotypes does not contradict its likely role as a positive regulator of dNTP levels. Introduction A largely conserved network of proteins is in place to prevent lethal and mutagenic consequences of DNA damage and replication stress in eukaryotes (Friedberg et al., 2006). In this context, the regulation of dNTP levels has attracted considerable attention and many details surrounding the regulation of the heterotetrameric ribonucleotide reductase (RNR) are known in Saccharomyces cerevisiae. RNR activity and level is increased in S-phase and in response to DNA damage or replicational stress (e.g. by hydroxyurea [HU]) through various mechanisms: by transcriptional regulation (inactivation of the Crt1 co-repressor), by subunit localization, by inhibitor interaction and degradation, and by altered feedback inhibition (Chabes et al., 2003; Elledge et al., 1993; Huang et al., 1998; Lee and Elledge, 2006; Wu and Huang, 2008; Yao et al., 2003; Zhao et al., 2001). In budding yeast, an elevated dNTP level can prevent the lethality of checkpoint kinase (Mec1 or Rad53) deletions. These checkpoint kinases are necessary for cell cycle arrest and facilitation of DNA repair (Friedberg et al., 2006; Nyberg et al., 2002). Elevated dNTP levels can be achieved by deletion of negative regulators such as Sml1, Ctr1 or Dif1 or by overexpression of RNR subunit Rnr1 (Desany et al., 1998; Lee et al., 2008; Zhao et al., 1998). Additional genes of unknown function have been identified through a selection for genes that, if overexpressed, suppress the lethality of a RAD53 and/or MEC1 deletion (srl , for suppressor of Rad53 lethality) (Desany et al., 1998). Some of these genes have remained uncharacterized. The present study concerns SRL3 whose deletion in a commercially available strain was identified by us in a screen for spontaneous mutator mutants. However, its high spontaneous mutability and DNA damage sensitivity phenotype were found to be associated with an independent mutation which was identified as a truncation of MMS2. MMS2 is involved in a replicative DNA damage tolerance pathway (Broomfield et al., 1998; Broomfield et al., 2001; Friedberg et al., 2006; Gangavarapu et al., 2006; Hofmann and Pickart, 1999; Torres-Ramos et al., 2002; VanDemark et al., 2001; Xiao et al., 1998). While we found no direct connection between SRL3 and MMS2, important information pertaining to both gene products was uncovered. Materials and Methods Yeast strains and strain constructionMost haploid strains were derived from BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0). The collection of deletions of non-essential genes made in this strain was purchased from OpenBiosystems. Additional deletions were introduced by replacing a specific gene with a PCR product containing micro-homology upstream and downstream of the gene and a selectable marker such as G418 resistance gene kanMX4 (Rothstein, 1989). Yeast transformation was performed by the lithium acetate method (Gietz and Schiestl, 1995). Sta